Recent advances in the field of molecular biology allow the production of transgenic animals (i.e., non-human animals containing an exogenous DNA sequence in the genome of germline and somatic cells introduced by way of human intervention). Differences in the regulation of these foreign genes in different cell types make it possible to promote the differential expression of the foreign gene in a preselected tissue, such as the mammary gland, for ease of isolation of the protein encoded by the foreign gene, for a desired activity of the foreign gene product in the selected tissues, or for other reasons.
An advantage of transgenic animals and differential gene expression is the isolation of important proteins in large amounts, especially by economical purification methods. Such proteins are typically exogenous to the transgenic animal and may comprise pharmaceuticals, food additives, nutritional supplements, and the like. However, exogenous proteins are preferably expressed in tissues analogous to those in which they are naturally expressed. For example, exogenous milk proteins (e.g., lactoferrin) are preferably expressed in milk-forming tissues in the transgenic animal. As a result, difficult isolation problems are presented because the exogenous protein is often expressed in the tissue or bodily fluid containing an endogenous counterpart protein (if it exists), and possibly other undesired contaminant species which may have very similar physicochemical properties. Moreover, many exogenous proteins must be substantially purified from other species, frequently purified to homogeneity, prior to their use as pharmaceuticals or food additives.
For example, the production of transgenic bovine species containing a transgene encoding a human lactoferrin polypeptide targeted for expression in mammary secreting cells is described in WO91/08216, incorporated herein by reference. The purification of human lactoferrin (hLF) from a transgenic animal containing a functional endogenous bovine lactoferrin (bLF) gene and a transgene encoding the expression of hLF is complicated by the presence of endogenous bLF which has physicochemical properties similar to human lactoferrin. Even in a transgenic bovine lacking a functional endogenous bLF gene (e.g., as a result of homologous gene targeting to functionally disrupt the endogenous bLF alleles), it is frequently desirable and/or necessary to purify transgene-encoded hLF from other biological macromolecules and contaminant species. Since hLF has potential pharmaceutical uses and may be incorporated in human food products as a nutritive supplement, uses which typically require highly purified hLF, it is imperative that methods be developed to purify hLF from milk, especially from milk or milk fractions of transgenic nonhuman animals such as bovine species.
Human lactoferrin is a single-chain glycoprotein which binds ferric ions. Secreted by exocrine glands (Mason et al. (1978) J. Clin. Path. 31: 316; Tennovuo et al. (1986) Infect. Immunol. 51: 49) and contained in granules of neutrophilic leukocytes (Mason et al. (1969) J. Exp. Med. 130: 643), this protein functions as part of a host nonspecific defense system by inhibiting the growth of a diverse spectrum of bacteria. hLF exhibits a bacteriostatic effect by chelation of the available iron in the medium, making this essential metal inaccessible to the microorganisms (Bullen et al. (1972) Brit. Med. J. 1: 69; Griffiths et al. (1977) Infect. Immunol. 15: 396; Spik et al. (1978) Immunology 8: 663; Stewart et al. (1984) Int. J. Biochem. 16: 1043). The bacteriostatic effect may be blocked if ferric ions are present in excess of those needed to saturate the hLF binding sites.
Lactoferrin is a major protein in human milk (present at a concentration of about 1.5-1.7 mg/ml) and may play a role in the absorption of dietary iron by the small intestine. Essentially all of the iron present in human breast milk is reported to be bound to hLF and is taken up at very high efficiency in the intestine as compared to free iron in infant formula (Hide et al. (1981) Arch. Dis. Child. 56: 172). It has been postulated that the efficient uptake of hLF-bound iron is due to a receptor in the jejunum. Specific lactoferrin receptors have been reported on mucosal cells of the small intestine of adult humans (Cox et al. (1979) Biochem. Biophys. Acta 588: 120).
hLF from human colostrum is available commercially (Calbiochem, La Jolla, Calif. and other vendors) as a lyophilisate for research applications in small amounts (10 mg and 25 mg vials). The amino acid sequence of hLF has been reported (Metz-Boutigue et al. (1984) Eur. J. Biochem. 1451: 659), and WO91/08216 reports an hLF sequence having some sequence inconsistencies with the previous report of Metz-Boutigue et al. hLF comprises two domains, each comprising an iron-binding site and an N-linked glycosylation site. These domains show homology with each other, consistent with an ancestral gene duplication and fusion event. hLF also shares extensive sequence homology with other members of the transferrin family (Metz-Boutigue et al. (1984) op.cit.; Pentecost et al. (1987) J. Biol. Chem. 262: 10134). A partial cDNA sequence for neutrophil hLF was published by Rado et al. (1987) Blood 70: 989), which agrees by more than 98% sequence identity compared to the amino acid sequence determined by direct amino acid sequencing from hLF from human milk. The structures of the iron-saturated and iron-free forms of human lactoferrin have been reported (Anderson et al. (1989) J. Mol. Biol. 209: 711; Anderson et al. (1990) Nature 344: 784).
Protocols for purifying lactoferrin from milk have been reported. U.S. Pat. No. 4,436,658 describes the isolation of bovine lactoferrin from defatted and casein-free whey of bovine milk. Briefly, whey is contacted with silica in a slightly basic medium at pH 7.7-8.8, the lactoferrin is adsorbed and thereafter eluted with 0.5M NaCl/0.1N acetic acid. U.S. Pat. No. 4,791,193 and European Patent Application No. EP 0 253 395 by Okonogi et al. similarly report a method wherein bovine milk is contacted with carboxymethyl groups of a weakly acidic cation exchange resin and the adsorbed lactoferrin is eluted with a 10 percent NaCl gradient. In U.S. Pat. No. 4,668,771, bLF is isolated from bovine milk using a monoclonal antibody fixed to an insoluble carrier. WO89/04608 describes a process for obtaining fractions of bovine lactoperoxidase and bLF from bovine milk serum; the milk serum is microfiltered and passed through a strong cation exchanger, pH&lt;6.0, at a high rate of flow for selective adsorption of lactoperoxidase and bLF followed by elution with a 0.5-2.0M NaCl solution. U.S. Pat. No. 4,997,914 discloses isolation of hLF from human colostrum or raw milk; the lactoferrin-containing sample is contacted with a sulfuric ester of a crosslinked polysaccharide to bind hLF, followed by elution with a 0.4-1.5 NaCl aqueous solution.
The scientific literature also reports protocols for the isolation of lactoferrin from milk. A number of these involve isolation of LF from a natural source using ion-exchange chromatography followed by salt elution. Querinjean et al. (1971) Eur. J. Biochem. 20: 420, report isolation of hLF from human milk on CM Sephadex C-50 followed by elution with 0.33M NaCl. Johannson (1969) Acta Chem. Scand. 23: 683 employed CM Sephadex C-50 for purification of LF, and Johannson et al (1958) Nature 181: 996 reports the use of calcium phosphate for LF purification. Torres et al. (1979) Biochem. Biophys. Acta 576: 385 report lactoferrin isolation from guinea pig milk. The milk was pre-treated by centrifugation to remove fats and to sediment the casein. A Whatman CM-52 column was used, and lactoferrin was eluted with 0.5M NaCl/5 mM sodium phosphate, pH 7.5. Roberts and Boursnell (1975) Jour. of Reproductive Fertility 42: 579, report lactoferrin isolated from defatted sow's milk. CM-Sephadex was added to an ammonium ferrous sulfate precipitate of the milk, and the bound lactoferrin was eluted with 0.5M NaCl/20 mM phosphate at pH 7 followed by a second CM-Sephadex fractionation from which the lactoferrin was eluted with 0.4M NaCl. Zagulski et al. (1979) Prace i Materialy Zootechniczne 20: 87, report bovine lactoferrin isolated from bovine milk. Defatted bovine milk was mixed with CM-Sephadex C-50, and lactoferrin was eluted from the column with 0.5M sodium chloride/0.02M sodium phosphate at pH 7. Moguilevsky et al. (1975) Biochem J. 229: 353, report lactoferrin isolated from human milk, using CM-Sephadex chromatography and elution with 1M sodium chloride. Ekstrand and Bjorck (1986) Jour. of Chromatography 358: 429, report lactoferrin isolated from human colostrum and bovine milk. Defatted bovine or human milk was acidified, adjusted to pH 7.8 and applied to a Mono S.TM. column. The bovine or human lactoferrin was eluted with a continuous salt gradient of 0-1M NaCl. The purification of human lactoferrin from bovine lactoferrin was not reported. Foley and Bates (1987) Anal. Biochem. 162: 296, report isolation of lactoferrin from human colostrum whey. The whey was mixed with a weak ion-exchange resin (cellulose phosphate) and proteins were eluted with a stepped salt and pH gradient. Lactoferrin was eluted with 0.25M NaCl/0.2M sodium phosphate at pH 7.5. Further, Yoshida and Ye-Xiuyun (1991) J. Dairy Sci. 74: 1439, disclosed the isolation of lactoferrin by ion exchange on carboxymethyl cation resin using 0.05M phosphate buffer at pH 7.7 with a linear gradient of 0-0.55M NaCl. The carboxymethyl-Toyopearl column adsorbed only lactoperoxidase and lactoferrin from the albumin fraction of bovine milk acid whey. Lactoferrin was eluted between 0.4-0.55M NaCl and was separated into two components; lactoferrin A and lactoferrin B.
Other methods, including affinity chromatography, have also been reported. For example, in Kawakami et al. (1987) J. Dairy Sci. 70: 752, affinity chromatography of LF with monoclonal antibodies to human or bovine lactoferrin was reported. Human lactoferrin was isolated from human colostrum and bovine lactoferrin from bovine milk or cheese whey. (See also U.S. Pat. No. 4,668,771, cited supra) Hutchens et al. (1989) Clin. Chem. 35: 1928, lactoferrin was isolated from the urine of human milk fed preterm infants with single-stranded DNA on an affinity column. Additionally, Chen and Wang (1991) J. Food Sci. 56: 701 reported a process combining affinity chromatography with membrane filtration to isolate lactoferrin from bovine cheese whey using heparin-Sepharose to bind lactoferrin. Cheese whey was diluted with a binding buffer and added to the heparin-Sepharose material. The slurry was microfiltered, and the lactoferrin was eluted with 5 mM veronal-hydrochloride/0.6M NaCl at pH 7.4. Bezwoda et al. (1986) Clin. Chem. Acta 157: 89 report the use of Cibacron Blue F3GA resin for purification of LF. Ferritin (Pahud et al. (1976) Protides Biol Fluids. 23: 571) and heparin (Blackberg (1980) FEBS Lett. 109: 180) have also been reported for purification from milk.
Thus there exists a need in the art for methods for purification of human lactoferrin from milk, particularly from milk of nonhuman transgenic animals, such as bovine species, that contain human lactoferrin encoded by a transgene. It is one object of the invention to provide methods and compositions for economical and efficient purification of human lactoferrin from milk, such as bovine milk, for use as a pharmaceutical or food additive. The present invention fulfills these and other needs. It is also an object of the present invention to provide human lactoferrin compositions with a purity of about 98% or greater.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.